1. Field of the Invention
The present invention relates to a method for drying electrophoresis gels and in particular is concerned with compositions and methods for drying polyacrylamide gels.
2. Background Art
Gel electrophoresis is a technique much used to separate biological macromolecules such as polypeptides, proteins, DNA and RNA. Polyacrylamide is one commonly used gel forming polymer.
In polyacrylamide gel electrophoresis, a polyacrylamide gel containing an appropriate buffer is cast into a thin slab between glass or plastic plates or enclosed in a glass tube. The slab is placed between electrode compartments and a small amount of a solution of the sample is pipetted into precast notches on the top of the gel. Usually a water soluble cationic or anionic tracking dye is added. The dye migrates faster than the macroions so that the progress of the experiment can be tracked. The current is turned on and run until the tracking dye band is near the bottom of the slab. The gel is then removed and generally stained with a dye that binds to the proteins or nucleic acids.
The gel cannot be stored for a prolonged period as it becomes brittle or swells excessively if left as is. Therefore, in many cases, profiles of electrophoresis gels are photographed or copied on paper or the like and stored as images.
However, in photographing or copying the gel it is difficult to accurately record thin bands or very subtle differences in density of stains. In addition, biological macromolecules which have been separated by electrophoresis cannot be recovered. In order to mitigate this problem various proposals for drying the gel for storage have been proposed.
U.S. Pat. No. 5,635,046 (Daiichi Pure Chemicals Co., Ltd.), the disclosure of which is incorporated herein by reference, describes prior art methods for drying gels.
One such method is dislosed in U.S. Pat. No. 4,883,597, the disclosure of which is also incorporated herein by reference. The patent describes a drying method in which water vapour is extracted from a gel supported by a polyethylene membrane by apply a vacuum through the membrane.
Other more complex methods involve drying devices utilizing both heat and vacuum sources in attempt to uniformly remove moisture from the gel. Such a procedure commonly involves positioning the gel matrix on a filter paper and placing the resulting combination of gel matrix on filter paper within a drying apparatus in which the combination is subjected to a vacuum source and heated to remove liquid from the gel. An example of such an arrangement is shown in U.S. Pat. No. 4,020,563. Similarly, U.S. Pat. Nos. 4,612,710 and 4,788,778 disclose a method and apparatus in which heat is provided to a horizontal gel slab from a heating plate below while drawing a vacuum beneath.
However, as pointed out in U.S. Pat. No. 5,635,046, a method in which gels are dried with heat under reduced pressure requires a dedicated drying apparatus and a vacuum pump, which are both relatively expensive. In addition, when gels contain acrylamides at high concentrations of not less than 15% by weight, frequently the gels are damaged due to cracking. Moreover, methods using an organic solvent to dewater gels tend to result in a loss in transparency or deformation of the gel, raising a problem in recording images accurately.
An alternative method for drying gels is to sandwich the gel between cellophane films (for example, extruded cellulose xanthate) or the like. An advantage of this method is that it does not require special equipment, apart from the optional use of a simple drying frame. Moreover, sandwiching the gel between two cellophane films has the further advantage of suppressing deformation of the gel during the drying process.
However, even when the cellophane sandwich method is used, cracking of the gel can occur, especially when using gels containing polyacrylamides at high concentrations of not less than 15% by weight.
Furthermore, the gels can lose transparency, particularly at low gel concentrations.
To avoid this problem, a number of methods have been proposed including incorporation of glycerol in the gel and application of gelatin or a paste onto the surface of a gel. However, these methods remain ineffective for preventing the occurrence of cracks in the case of gels having high concentrations of acrylamide.
The present inventor has found that the use of polyhydroxy alcohols in the drying process gives transparent gels that do not experience significant cracking during the dry process, even where the gels have high or low concentrations of polyacrylamide. This is a surprising outcome give that the use of glycerol, a polyol, results in significant cracking of the gel during the drying process.
Without wishing to limit the present invention in any way, it is believed that the gel cracking resulting from the use of glycerol in the drying process is in some way related to the vicinal nature of three hydroxy groups of glycerol (1,2,3 propane triol). The inventor has found that cracking of the gel during the drying process can be avoided provided that the polyhydroxy alcohol used has no vicinal hydroxy groups, or if it does have vicinal hydroxy groups, it has no more that 2 vicinal hydroxy groups.
Accordingly, the present invention provides a method for drying a polyacrylamide gel, the method comprising contacting the gel with au aqueous solution of a polyhydroxy alcohol other than a polyhydroxy alcohol having at least 3 vicinal hydroxy groups and drying the gel.
The polyacrylamide gel to be dried according to the method of the present invention is not particularly limited as long as it can be used for electrophoresis. For example, it may be a polyacrylamide gel having an arbitrary concentration or density gradient ranging from about 2 to 50% by weight. The polyacrylamide gel may be that formed from a substituted or unsubstituted acrylamide and optionally one or more other monomers.
The polyhydroxy alcohol used in the method of the invention may be any alcohol having 2 or more hydroxy groups provided that the alcohol is not one having 3 or more vicinal hydroxy groups. Preferably the polyhydroxy alcohol has a ratio of carbon to hydroxy groups such that the polyhydroxy compound remains water soluble. More preferably, the polyhydroxy alcohol has no more than 6 hydroxy groups. For example, the polyhydroxy alcohol may be a diol, triol or tetraol. Particular examples of suitable polyhydroxy alcohols are alkyl diols and alkyl tetraols. Particular example of polyhydroxy alcohols include 1,2-ethane diol, 1,2-propane diol, 1,3propane diol 1,4-butane diol, 1,6 hexane diol, 1,2,6-trihydroxy hexane, trimethylol propane and pentaerythritol. The polyhydroxy alcohol may be a polyglycerol, which is formed by the self-condensation of glycerol. The ether linkage between the glycerol units in the polyglycerol may be 1, 2 or 1, 3.
Surprisingly, it has been found that polyhydroxy alcohols in accordance with the present invention can be used in the method of the invention as an aqueous solution, without the need for an additional solvent. Not having to use additional solvents has obvious advantages in terms of environmental and safety issues. Although not required, a solvent other than water may be included in the aqueous solution. The other solvent may be any solvent used in polyacrylamide gel electrophoresis. The solvent may be such that it alters the rate of drying of the gel. The other solvent may be a water-soluble and highly volatile organic solvent. Examples of such organic solvents include C1-C4 alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and butanol, as well as solvents such as acetone, tetrahydrofuran, acetonitrile, dimethyl formamide and dimethylsulfoxide. In the present invention, methanol, ethanol, 1-propanol, and 2-propanol are preferred. The organic solvents may be used singly or in combination of two or more. It is preferred that the organic solvents be present in amounts of 0.1-80% by weight, and more preferably 1-50% by weight, in the aforementioned aqueous solution.
The polyhydroxy alcohol may be present in an amount ranging from about 0.1% to about 20% of the aqueous solution. The actual concentration of the polyhydroxy alcohol in the aqueous solution depends on the particular polyhydroxy alcohol used Ed the nature of the solveuts used in the solution.
The aqueous solution containing the polyhydroxy alcohol may include other components conventionally used in gel drying solutions. For example, the aqueous solution may contain one or more viscosity control agents, for example, a water soluble polymer.
In the method of the present invention, the solution component contained in a polyacrylamide gel following electrophoresis may be replaced by the aqueous solution of the polyhydroxy alcohol by immersing the gel in the aqueous solution for at least 1 minute end preferably for 10 minutes to 24 hours. When the aqueous solution is stirred or shaken, the time required for replacement of the components in a polyacrylamide gel can be reduced. The volume of the aqueous solution is preferably at least equivalent to that of the polyacrylamide gel to dry, and particularly preferably at least 4 times that of the gel. The polyacrylamide gel used for electrophoresis is preferably washed with water or the like before being immersed in the aqueous solution contain the polyhydroxy alcohol.
The drying step of the method of the present invention may be according to any of the prior art drying methods described above The drying method used in the method of the present invention may be air drying or vacuum assisted drying. Preferably the gel is dried by placing the gel, which has been treated with an aqueous solution in accordance with the present invention, between two sheets or films at least one of which is a porous hydrophilic film. The porous hydrophilic sheet or film may be a cellophane film. Most preferably, the gel is dried by sandwiching it between two cellophane films held between two drying frames. Drying frames are well known. The drying frames may simply be rectangular frames made of a suitable substance, for example, perspax. The drying frames may be a more complex arrangement, for example, that described in U.S. Pat. No. 5,572,802, the disclosure of which is incorporated herein by reference. The two frames may be clamped together by using one or more clamps, for example, bulldog clips.
Throughout this specification the word comprised or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
In order that the present invention may be more readily understood, the following non-limiting embodiments are provided.